CELL-SPECIFIC TRANSCRIPTIONAL REGULATION AND REACTIVATION OF GALECTIN-1 GENE-EXPRESSION ARE CONTROLLED BY DNA METHYLATION OF THE PROMOTER REGION

The galectin-1 gene is a developmentally regulated gene whose activity is strongly modulated during cell differentiation and transformation. We have previously shown that galectin-1 promoter constructs are high ly active when transiently transfected in cells both expressing and no t expressing the endogenous gene and that the basal activity is determ ined by a small region encompassing the transcription start site (from positions -50 to +50). We have now investigated the role of DNA methy lation in galectin-1 gene expression. Southern blot analysis with HpaI I and MspI endonucleases and sodium bisulfite analysis of genomic DNA from expressing and nonexpressing cell lines and cell hybrids showed a close correlation between gene activity and demethylation of the 5' r egion of the galectin-1 gene. We found that the galectin-1 promoter re gion is fully methylated, at every CpG site on both strands, in nonexp ressing differentiated rat liver (FAG) and thyroid (PC Cl3) cells and unmethylated in the expressing undifferentiated liver (BRL3A) and thyr oid transformed (PC myc/raf) cell lines. In addition, reactivation of the silent FAG alleles in the FAG-human osteosarcoma (143TK(-)) hybrid cells is accompanied by a complete demethylation of the promoter regi on. Finally, when galectin-1-chloramphenicol acetyltransferase (CAT) p romoter constructs were methylated in vitro by SssI methylase at every cytosine residue of the CpG doublets and transfected into mouse fibro blasts, the transcription of the CAT reporter gene was strongly inhibi ted.